Nkt-cell subset for in vivo persistence and therapeutic activity and propagation of same

ABSTRACT

Embodiments of the disclosure include methods and compositions for producing NKT cells effective for immunotherapy and also methods and compositions for providing an effective amount of NKT cells to an individual in need of immunotherapy. In specific embodiments, the NKT cells are CD62L+ and have been exposed to one or more costimulatory agents to maintain CD62L expression. The NKT cells may be modified to incorporate a chimeric antigen receptor, in some cases.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application is a Continuation of U.S. Non-provisionalapplication Ser. No. 15/135,453 filed 21 Apr. 2016; which claims thebenefit of U.S. Provisional Application No. 62/309,525 filed 17 Mar.2016 and U.S. Provisional Application No. 62/151,690 filed 23 Apr. 2015;each of which are incorporated herein by reference in their entirety forall purposes.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under grant numbersCA116548 and CA126752 awarded by the National Institutes of Health. Thegovernment has certain rights in the invention.

TECHNICAL FIELD

Embodiments of the disclosure encompass at least the fields of cellbiology, molecular biology, immunology, and medicine, including at leastcancer medicine.

BACKGROUND

Type-I NKT cells (NKTs) are an evolutionary conserved subset of innatelymphocytes that express invariant TCRα-chain Vα24-Jα18 and react toself- or microbial-derived glycolipids presented by monomorphic HLAclass-I like molecule CD1d (Porcelli et al. (1993); Lantz and Bendelac,1994; Bendelac et al., 1995; Kim et al., 2015). The potential importanceof NKTs for tumor immunity and immunotherapy has been demonstrated inmultiple models of cancer in mice and in early stage clinical trials incancer patients (McEwen-Smith et al., 2015; Dhodapkar, 2009; Exley andNakayama, 2011; Motohashi et al., 2011; Yamasaki, 2011; Taniguchi etal., 2015). In contrast to T cells, NKTs effectively traffic to thetumor site and can mediate anti-tumor responses either via directkilling of CD1d+ tumor cells, inhibition of tumor-supportivemacrophages, or trans-activation of NK cells (Metelitsa, 2011). Severalstudies have revealed strong positive associations between the numbersof tumor-infiltrating or circulating NKTs and improved disease outcomein patients with diverse tumor types (Dhodapkar, 2009; Metelitsa et al.,2004; Tachibana et al., 2005; Molling et al., 2007; Cariani et al.,2012; Cariani et al., 2012). Conversely, tumor progression is oftenaccompanied by a decrease in NKT-cell number or functional activity(16), or the downregulation of CD1d expression on malignant cells(Dhodapkar et al., 2003). To counteract these tumor escape mechanisms,methods were developed to expand primary human NKTs to clinical scale exvivo and to redirect their cytotoxicity against tumor cells viatransgenic expression of chimeric antigen receptors (CARs) (Heczey etal., 2014). Similar to the observations reported in CAR T-cell clinicaltrials (Kalos and June, 2013; Dotti et al., 2014), there is a strongcorrelation between the anti-tumor efficacy and in vivo persistence ofCAR NKT-cell products in a xenogenic tumor model (Heczey et al., 2014).However, the mechanisms that govern ex vivo expansion and subsequent invivo persistence of human NKTs remain largely unknown, impeding rationaldesign of NKT cell-based cancer immunotherapy.

Recent global transcriptional profiling studies demonstrated that NKTs,though they share properties with T and NK cells, are a distinctpopulation of lymphocytes (Cohen et al., 2013). In the mouse, thedevelopmental program and functional differentiation of NKTs have beencharacterized quite extensively during the last decade, as summarized inrecent reviews (Kim et al., 2015; Contantinides and Bendelac, 2013).Several key features of murine NKTs have also been confirmed in theirhuman counterparts. Both in mice and humans, NKTs diverge from T cellsat the stage of CD4+ CD8+ (double positive, DP) thymocytes. Unlike Tcells, which are positively selected by thymic epithelial cells, NKTsare selected by CD1d-expressing DP thymocytes (Gapin et al., 2001). Theexpression of promyelocytic leukemia zinc finger transcription factor(PLZF) immediately after positive selection enables intrathymicexpansion and effector/memory-like differentiation of NKTs (Savage etal., 2008). Peripheral NKTs are long-lived lymphocytes and theirpost-thymic maintenance largely depends on slow IL-15-mediatedhomeostatic proliferation (Matsuda et al., 2002; Baev et al., 2004). Inhuman peripheral blood, NKTs are divided into two major functionalsubsets based on CD4 expression: CD4+ and CD4− (mostly CD8/CD4-doublenegative, DN) (Lee et al., 2002). The CD4+ subset is highly enriched inneonate NKTs and undergoes fewer homeostatic divisions compared with theCD4− subset in adults (Baev et al., 2004), suggesting that CD4+ NKTscould contribute to the long-term persistence of adoptively transferredtherapeutic NKTs under certain conditions. However, ex vivo expansion ofhuman NKTs in response to antigenic stimulation, e.g., withαGalactosylceramide (αGalCer), produces similar numbers of CD4+ and DNNKTs (28). NKTs also exhibit an NK-like linear differentiation withacquisition of CD161 and then CD56 expression. Like in T cells, theexpression of CD56 is associated with terminal differentiation and theloss of proliferative potential (Loza et al., 2002).

In contrast to peripheral T cells, which have a well-establisheddevelopmental hierarchy from naïve to central memory to effector memoryto terminal effector cells (Sallusto et al., 2004), NKTs are broadlydescribed as cells with “activated/memory” phenotype without the naïvestate (D'Andrea et al., 2000; Kronenberg and Gapin, 2002). In cordblood, the majority of NKTs are CD4+ and co-express CD45RO with CD62Land CCR7 without immediate effector function (Baev et al., 2004;D'Andrea et al., 2000; Eger et al., 2006), thus resemblingcentral-memory CD4 T cells. In adult peripheral blood, NKTs are equallysplit between CD4+ and CD4− subsets (albeit with significantinter-individual variability). Adult NKTs lack a clear demarcationbetween “memory” and “effector” states, as they variably express memorymarkers and have immediate effector functions such as cytokineproduction and cytotoxicity (Baev et al., 2004; Eger et al., 2006). Themajority of adult NKTs even in the elderly express CD28 (DelaRosa etal., 2002), making them distinct from terminally differentiated Teffector cells (Okada et al., 2008).

Recent reports have demonstrated that CD62L+ central memory T cells havestem cell properties and superior therapeutic activity in cell therapyproducts (Graef et al., 2014; Wang et al., 2012; Sommermeyer et al.,2015). The functional significance of CD62L expression in NKTs remainsunknown. In this disclosure, the CD62L+ subset is required for NKT cellex vivo expansion and in vivo persistence. Importantly, when engineeredto express CD19-specific CAR (CAR.CD19), CD62L+ but not CD62L− CAR.CD19NKTs produced sustained tumor regression in a B-cell lymphoma model inNSG mice. CD62L+ NKTs could be maintained during ex vivo expansion whenprovided with certain costimulatory ligands. With this knowledge, onecan engineer co-stimulatory artificial antigen-presenting cells (aAPC)that can be used to generate NKTs and CAR-NKTs with superior therapeuticactivity in patients with cancer, for example.

BRIEF SUMMARY

Methods and compositions of the present disclosure concernimmunotherapies for an individual in need thereof. In some embodiments,the individual is in need of a therapy that targets a particularantigen-bearing cell for destruction, such as cancer, for example. Thedisclosure generally provides for the use of NKT cells for immunotherapybased upon improvements of methods to generate clinically useful amountsand efficacies of NKT cells.

Embodiments of the disclosure provide CD62L+ NKT cells that havesuperior in vivo persistence and anti-tumor activity. Embodiments of thedisclosure allow for effective expansion of NKT cells such that they canbe used for therapeutic applications. NKTs of the present disclosurehave enhanced survival and expansion that are associated with theexpression of CD62L. The expression of CD62L is present in the NKT cellsand is maintained in the cells because of co-stimulation of the NKTcells. Embodiments of the disclosure include co-stimulation of NKT cellsby any method to maintain CD62L expression. The NKT cells are exposed toco-stimulation using one or more methods, such as upon exposure to oneor more cytokines (including at least IL-21), one or more agonisticantibodies that bind to a costimulatory receptor, and/or artificialantigen presenting cells that express CD1d and one or more costimulatoryreceptor ligands, for example. Thus, in specific embodiments one canutilize artificial antigen-presenting cells for the generation ofCD62L-enriched NKTs for effective cancer immunotherapy.

In one embodiment, there is a method of preparing natural killer T (NKT)cells for use in immunotherapy, comprising the step of enriching apopulation of NKT cells for CD62L-positive NKT cells. In a specificembodiment, the CD62L-positive NKT cells are activated by stimulation ofT-cell receptor and co-stimulation by costimulatory receptors and/orcytokines. In some cases, the method further comprises the step ofdelivering a therapeutically effective amount of the cells to anindividual in need of therapy. In certain aspects, the cells aremodified to express one or more chimeric antigen receptors, T-cellreceptors, one or more cytokines, one or more cytokine receptors, one ormore chimeric cytokine receptors, or a combination thereof.

In a certain embodiment, there is a method of treating an individual fora medical condition using immunotherapy, comprising the steps of a)enriching a population of NKT cells for CD62L-positive NKT cells orobtaining a population of NKT cells that are enriched for CD62L-positiveNKT cells; and b) providing a therapeutically effective amount of theCD62L-positive NKT cells to the individual.

In an embodiment, there is a method of treating an individual for amedical condition using immunotherapy, comprising the steps of expandingCD62L+ NKT cells from a population mixture of CD62L+ NKT cells andCD62L− NKT cells by exposing the population mixture to one or moreco-stimulatory agents to enrich for and produce co-stimulated CD62L+ NKTcells; and providing a therapeutically effective amount of theco-stimulated CD62L+ NKT cells to the individual. In specificembodiments, stimulatory agents and the co-stimulatory agents comprisea) one or more cytokines; b) a substrate that comprises an agonisticantibody or ligand for T-cell receptor (e.g., OKT3 mAb, 6611 mAb, orrecombinant human CD1d with bound agonistic glycolipid such asalpha-galactosylceramide) and one or more agonistic antibodies thattarget co-stimulatory receptors; or c) an antigen presenting cell thatcomprises CD1d expression and that comprises expression of one or moreligands of one or more costimulatory receptors. In certain embodiments,the cytokine is selected from the group consisting of IL-21, IL-2, IL-7,IL-15, IL-12, TNFalpha, and a combination thereof. The substrate may bea bead, plate, or a gel. In a specific embodiment, the antigenpresenting cell is transduced with one or more polynucleotides toexpress one or more ligands of one or more co-stimulatory receptors. Theco-stimulatory receptor may be CD28, OX40, 4-1BB, ICOS, CD40, CD30,CD27, or a combination thereof. The ligand of the co-stimulatoryreceptor may be CD80, CD86, OX4OL, 4-1BBL, ICOS ligand, CD154, CD3OL, ora combination thereof.

In particular embodiments, NKT cells encompassed by the disclosurecomprise a genetic modification. In specific aspects, the geneticmodification provides the cells with cancer cell-targeting activity,such as targeting of an antigen on cancer cells. The geneticmodification may comprise a T-cell receptor and/or a chimeric antigenreceptor. In some cases, the NKT cells are genetically modified afterthe exposing of the population to one or more co-stimulatory agents. TheNKT cells may be genetically modified within 1, 2, 3, 4, 5, 6, or moredays after the exposing of the population to one or more co-stimulatoryagents.

In a certain embodiment, there are methods of producing NKT cells forimmunotherapy, comprising the step of costimulating a population of NKTcells to maintain CD62L expression in at least some of the NKT cells. Insome cases, the methods further comprise the step of providing aneffective amount of the NKT cells to an individual in need thereof.

In one embodiment, there is a method of producing NKT cells forimmunotherapy, comprising the step of exposing a pre-sorted populationof CD62L+ NKT cells or a mixed population of CD62L+ NKT cells and CD62L−NKT cells to one or more co-stimulatory agents designed to purposelyenrich or retain a population of CD62L+ NKT cells. In some cases, themethod further comprises the step of obtaining the mixed population. Inspecific embodiments, the mixed population is from an individual towhich the enriched population will be delivered. In certain aspects, themixed population is from an individual that is different from theindividual to which the enriched population will be delivered. The mixedpopulation may be from a depository or obtained commercially.

In one embodiment, there is a composition of matter that comprises anon-natural cell expressing CD1d and expressing one or more ligands ofone or more co-stimulatory receptors.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1D. Accumulation of CD62L+ subset in culture after in vitroantigenic stimulation of primary NKTs. (1A) CD62L expression wasexamined by FACS in primary NKTs from freshly isolated PBMCs (day 0) and12 days after stimulation with αGalCer and in vitro expansion inculture. (1B) Kinetics of CD62L expression in NKTs at the indicatedintervals after primary stimulation (as in 1A) from individual donors(n=10). (1C) Expression of PD-1, TIM-3 and LAG-3 on NKT-cell surface wasmeasured by FACS on days 0 and 12. Representative plots from one of 4donors (upper panel) or Mean±SD of MFI for all donors (lower panel).(1D) On day 12 after primary stimulation, NKTs were magnetically sortedinto CD62L+ and CD62L− subsets followed by RNA isolation and geneexpression analysis using Human Immunology Panel v.2 and nCounterAnalysis System. The heat map shows the 1og2 fold changes (CD62+/CD62L−) of genes with p-values less than 0.02 and average fold changegreater than 2. Data were generated from 6 NKT-cell donors (12 pairedsamples).

FIGS. 2A-2E. Functional characterization of CD62L+ and CD62L− NKTs. (2A)Luciferase-transduced CD1d+ DAOY cells were pulsed with PBS (control) orαGalCer overnight followed by co-culture with CD62L+ or CD62L−magnetically sorted NKTs. Cytotoxicity was analyzed after 4 h bymeasuring luminescence intensity with a plate reader. Left plot is arepresentative of 3 donors with no difference in cytotoxicity betweenNKT subsets. Right plot is a representative of 3 donors with asignificant difference in cytotoxicity between NKT subsets. (2B) Theconcentrations of IFN-γ and IL-4 were measured in 24-h supernatants ofαGalCer-stimulated CD62L+ or CD62L− NKTs by the Luminex assay in 3independent experiments with NKTs from 3 donors. (2C) CFSE-labeled NKTswere magnetically sorted into CD62L+ and CD62L− subsets as confirmed bypost-sorting FACS (top plot) and stimulated with irradiatedαGalCer-pulsed APC. On day 3 after stimulation, staining for Annexin Vand 7-AAD was analyzed in NKTs by FACS after gating on CFSE-positiveevents. Results are from a representative of 5 donors tested (middlepanel). The corresponding bar graph (lower panel) shows Mean±SD ofpercent Annexin V+ NKTs on day 3 (N =5). (2D) Cell proliferation wasassessed on day 6 after stimulation as measured by CFSE dilution.Results are from a representative of 5 donors tested (upper panel) andMean±SD of CFSE MFI for all 5 donors (lower panel). (2E) Total cellcounts were performed at the indicated time intervals after NKT-cellstimulation. Shown are Mean±SD viable cells for a representative donor(upper panel) or fold change for each of 5 donors tested on day 6 afterstimulation. ***P<0.001, paired t-test.

FIGS. 3A-3D. CD62L+ NKTs have superior in vivo persistence andanti-tumor activity. (3A) Luciferase-transduced NKTs were sorted intoCD62L+ and CD62L− subsets and injected to NSG mice. NKT-cell in vivopersistence was monitored with bioluminescence imaging. (3B) Mean±SD ofbioluminescence photon count on indicated days after injection of CD62L+or CD62L− NKTs (P=0.008, paired t-test). (3C) Each mouse received i.v.injection of 2×10⁵ luciferase-transduced Daudi lymphoma cells (day 0)followed by (day 4) i.v. injection of 10⁷ CAR.CD19-transduced NKTs withIL-2 (1000 U/mouse) or PBS as a control. Tumor growth was monitoredusing bioluminescent imaging once per week. (3D) Survival probabilitywas analyzed by the Kaplan-Meier method (10 mice per group). Thedifferences in survival were then compared using the Log-rank test.

FIGS. 4A-4D. Co-stimulation maintains CD62L+ NKTs and preventsexhaustion. (4A) NKTs were sorted into CD62L+ and CD62L− subsets andstimulated and examined for expression of 4-1BB and OX40 by FACS beforeand 3 days after stimulation with αGalCer. Shown are plots from arepresentative of 4 donors. (4B) CD62L+ NKTs were stimulated on platescoated with the indicated agonistic mAbs. Shown are Mean±SD (N=4) offold change in absolute NKT-cell number on day 7 after stimulationcompared to day 0. P<0.001, one-way ANOVA. (4C) CD62L+ NKTs werestimulated as in B and analyzed for CD62L expression (black) vs. isotypecontrol (grey) on day 7. Shown are representative overlay histograms(upper panel) and Mean±SD of percent CD62L+ cells, N=4. (4D) CD62L+ NKTswere stimulated as in B and analyzed for the expression of PD-1 (black)vs. isotype control (grey) on day 12. Shown are representative overlayhistograms (upper panel) and Mean±SD of percent PD-1+ cells. ** or***P<0.01 or 0.001, one-way ANOVA.;

FIGS. 5A-5E. Phenotypic analysis of freshly isolated and in vitroexpanded NKTs. (5A) CD4 and CD62L expression was examined by FACS inprimary NKTs (gating on CD3+ Vα24-Jα18+ subset) in freshly isolated cordblood mononuclear cells (CBMC). Plots are from a representative of 5CBMC donors. (5B) CD4 and CD62L expression was examined in primary NKTsafter gating as in A before (day 0) and 12 days after stimulation withαGalCer and in vitro expansion. Plots are from a representative of 10PBMC donors. (5C) Expression of CCR7, CD27 and CD28 in relationship toCD62L expression in primary NKTs after gating as in A before (day 0) and12 days after stimulation with αGalCer and in vitro expansion. Plots arefrom a representative of 6 PBMC donors. (5D) Expression of CD161, CD56and IL7Rα in relationship to CD62L expression on day 12 afterstimulation with αGalCer and in vitro expansion. Plots are from arepresentative of 3 PBMC donors. (5E) Expression of PLZF, LEF1, andGATA3 in relationship to CD62L expression and co-expression of LEF1 andGATA3 was analyzed using intracellular flow cytometry on day 12 afterstimulation with αGalCer and in vitro expansion. Plots are from arepresentative of 4 PBMC donors.

FIGS. 6A-6B. The comparison of NKT-cell purity and absolute numbersafter expansion with CD3/CD28 agonistic mAbs vs. αGalCer-pulsedirradiated PBMC. NKTs were isolated from four PBMCs. Half of them werestimulated using autologous irradiated PBMC pulsed with αGalCer, anotherhalf were stimulated with CD3/CD28 mAb-coated plate. In both cases,cells were propagated in culture with the IL-2 (200 U/ml) added everyother day. At day 12, cultures were analyzed for: (6A) NKT-cell puritywas determined by flow cytometry as percent cells expressing CD3 andiTCRα. (6B) NKT-cell absolute cell count was performed using trypan blueexclusion assay in triplicates. *P<0.05, data were analyzed after Log(2)transformation using paired t test.

FIGS. 7A-7B. NKT-cell transduction with CAR.CD19. (7A) Schematicpresentation of CAR.CD19 construct. (7B) NKTs were re-stimulated withautologous PBMC (irradiated with 40 Gy). On day 3 after re-stimulation,24 well, non-tissue culture plates were coated with retronectin andafter washing inoculated with 1 ml of retroviral supernatant containingCAR.CD19. The viral supernatant was then removed and NKTs were added tothe wells in complete media and 200 U/ml rhIL-2. NKTs were thenmagnetically sorted to CD62L+ and CD62L− subsets and CAR.CD19 surfaceexpression was analyzed with 2D3 mAb staining by FACS 12 days aftertransduction. Shown are FACS plots from a representative of 3independent experiments.

FIGS. 8A-8B. Expression of co-stimulatory receptors on resting andactivated NKTs. (8A) FACS analysis of OX40 and 4-1BB expression inrelation to CD4 on resting NKTs (day 12 after primary stimulation) and 3days after re-stimulation with αGalCer. Plots are from a representativeof 6 PBMC donors. (8B) Magnetically sorted CD62L+ and CD62L− NKTs wereanalyzed for OX40 and 4-1BB expression in relation to CD4 3 days afterNKT cell restimulation with αGalCer. Plots are from a representative of4 PBMC donors.

FIG. 9. The comparison of NKT-cell expansion using low and highconcentrations of plate-bound OKT3 mAb. In vitro expanded quiescent NKTswere stimulated with anti-CD3 OKT3 mAb at 20 ng/ml or 1 μg/ml alone orwith 500 ng/ml anti-CD28 CD28.2 mAb. Cells were propagated in culturewith the IL-2 (200 U/ml) added every other day. At day 12, NKT-cellabsolute cell count was performed using trypan blue exclusion assay intriplicate and divided to the input number at day 0. Data are M SD, N=4.**P=0.01, paired t test.

FIGS. 10A-10C show that IL-21 increases frequency of CD62L+ NKT cellsduring primary expansion.

FIGS. 11A-11C demonstrate that IL-21 increases frequency of CD62L+ NKTcells during secondary expansion.

FIG. 12 shows expression of CD1d and co-stimulatory molecules on Ramoscells, as an example.

FIGS. 13A-13C demonstrate that Ramos cells can expand primary NKTs withhigh-level CD62L expression.

FIG. 14 shows that Ramos cells expand NKTs upon secondary stimulationwith significant retention of CD62L expression.

DETAILED DESCRIPTION

As used herein the specification, “a” or “an” may mean one or more. Asused herein in the claim(s), when used in conjunction with the word“comprising”, the words “a” or “an” may mean one or more than one. Asused herein “another” may mean at least a second or more. In specificembodiments, aspects of the invention may “consist essentially of” or“consist of” one or more sequences of the invention, for example. Someembodiments of the invention may consist of or consist essentially ofone or more elements, method steps, and/or methods of the invention. Itis contemplated that any method or composition described herein can beimplemented with respect to any other method or composition describedherein. The scope of the present application is not intended to belimited to the particular embodiments of the process, machine,manufacture, composition of matter, means, methods and steps describedin the specification.

I. General Embodiments

The disclosure provides NKT cells suitable for use in immunotherapybecause they are able to be expanded to sufficient levels and persist invivo at sufficient levels to achieve a therapeutic effect. The NKT cellsof the present disclosure are manipulated to express and maintainexpression of CD62L that at least in part allows them to have enhancedtherapeutic applicability. Such preservation of expression of CD62L inthe NKT cells occurs at least in part upon costimulation, including uponexposure to one or more costimulatory agents.

II. NKT Cells and Costimulation Thereof

In particular embodiments, NKT cells are useful for therapeuticapplication because they have enhanced in vitro expansion and in vivopersistence following exposure to one or more costimulatory agents thatallowed the cells to maintain CD62L expression. The one or morecostimulatory agents may be of any kind, but in specific embodimentsthey comprise one or more cytokines; b) a substrate (bead, plate, and soforth, for example) that comprises one or more agonistic antibodies thattarget co-stimulatory receptors; and/or c) a cell, such as an antigenpresenting cell, that comprises CD1d expression and that comprisesexpression of one or more ligands of one or more costimulatoryreceptors. In cases wherein the NKT cells are exposed to cytokines, thecytokines may be of any suitable kind, but in specific cases thecytokine is IL-21, IL-2, IL-7, IL-15, IL-12, IL-18, TNFalpha, or acombination thereof. In specific embodiments, the CD62L+ NKT cellsexpress IL-21 receptor and upon culture in the presence of IL-2 andIL-21 retain CD62L expression.

wherein the NKT cells are exposed to one or more costimulatory agentsthat are agonistic antibodies (in at least some cases that aremonoclonal) that immunologically recognize a co-stimulatory receptor,the receptor may be any co-stimulatory receptor. However, in specificembodiments the receptor is CD28, 4-1BB, OX-40, ICOS, CD2, CD27, CD30,GITR, TIM1, LFA1, ICAM1, or HVEM, for example. The antibodies may beaffixed to a substrate that allows a population of cells, such as apopulation of NKT cells, to be sufficiently exposed to the antibodies.The antibodies may be obtained commercially, obtained as a gift, orproduced by standard means in the art.

In cases wherein the NKT cells are exposed to a therapeuticallyeffective amount of cells that have antigen presenting cell activity,such as an artificial antigen presenting cell (such as a non-naturalcell with antigen presenting cell activity), the cell may be transducedwith one or more polynucleotides to express one or more ligands of oneor more costimulatory receptors. The cell may be of any kind so long asit expresses one or more ligands of one or more costimulatory receptors,but in at least some cases the cell expresses CD1d also. The cell is anantigen presenting cell, in particular embodiments. In specific cases, acell that expresses CD1d and/or that expresses one or more ligands ofone or more costimulatory receptors is naturally occurring and may beused in the methods encompassed herein. In other cases, a cell thatnaturally does not naturally express CD1d and/or that does not naturallyexpress one or more ligands of one or more costimulatory receptors istransduced to express the respective component(s) and is used in methodsencompassed herein. The ligand of the costimulatory receptor may be ofany kind, but in specific embodiments the ligand is CD80, CD86, 4-1BBL,OX40L, ICOSL, CD30L, GITRL, TIM4, LIGHT, and so forth. In cases whereinthe cell that has antigen presenting cell activity is transduced withone or more polynucleotides, the polynucleotides may be comprised on avector, including a viral vector or non-viral vector (such as aplasmid). Viral vectors include retroviral vectors, lentiviral vectors,adenoviral vectors, adeno-associated viral vectors, and so forth. Thevectors will comprise suitable regulatory element(s) for expression inthe cell that has antigen presenting cell activity. In some cases thepolynucleotide transduced into the cell that has antigen presenting cellactivity encodes two or more coding regions, such as encoding twocostimulatory receptor ligands. In such a case, the separate codingregions may or may not be regulated by the same regulatory element(s).

In embodiments wherein expression of CD62L on NKT cells is sustainedbecause of co-stimulation of the NKT cells, there may or may not be ageneral order to the steps for preparing the desired NKT cells. Inparticular embodiments, NKT cells are obtained from an appropriatesource (for example, blood (including peripheral blood, cord blood, andso forth)), by sorting (e.g., magnetic bead or FACS sorting) and arepresent in a mixed population of cells. Following this, the NKT cellsare activated via TCR stimulation using a substrate containing agonisticantibody or ligand for T-cell receptor (e.g., OKT3 mAb, 6611 mAb, orrecombinant human CD1d with bound agonistic glycolipid such asalpha-galactosylceramide) or antigen-presenting cells expressing CD1dand bound agonistic glycolipid such as alpha-galactosylceramide.TCR-activated NKT cells may be exposed to co-stimulation to producehigher levels of CD62L± NKT cells from the population than would occurin the absence of co-stimulation. In some embodiments, prior to deliveryto an individual in need thereof and following exposure to one or morecostimulatory agents, the NKT cells are manipulated by recombinant meansto incorporate one or more characteristics, such as to express one ormore therapeutic agents or entities that render the NKT cellstherapeutic. In certain embodiments, the cells are genetically modifiedto provide them with the ability to target an antigen-bearing cell. Inspecific embodiments, the manipulation is to transduce the NKT cells toexpress one or more chimeric antigen receptors and/or a T-cell receptor,either or which target a specific antigen of interest. In specificembodiments, the antigen is a tumor antigen.

NKT cells to be utilized for treatment of a medical condition in anindividual may originate from the individual to which they will beadministered, they may originate from another individual, or they may beobtained from a cell depository. The NKT cells are type 1 NKT cells, inspecific embodiments.

NKT cells may or may not be sorted prior to delivery to an individual.In specific embodiments, CD62L-positive NKT cells are not sorted fromCD62L-negative NKT cells, but in alternative embodiments they may besorted. In cases where cells are not sorted, for example based onwhether or not they express CD2L, when the cells are not sorted byphysical separation they can be enriched using co-stimulation of thecells that results in maintenance of CD62L expression.

In most cases the cells are not sorted based on a particular phenotype,but in some cases where cells are sorted, they may do so by methods thatallow enrichment of the desired cells, such as by collecting the desiredcells upon exposure to one or more substrates that are able tospecifically bind the cells. For example, one may utilize separation ofthe desired cells using an antibody on a substrate (such as a bead,particle, plate, gel matrix, and so forth), wherein the antibody maydirectly or indirectly bind the cells. In specific embodiments, magneticseparation may be employed.

III. Genetic Modification of NKT Cells

In particular embodiments, the NKT cells are genetically modified priorto delivery to an individual in need thereof. NKT cells are usuallymodified after TCR-stimulation and co-stimulation; in particularembodiments the genetic modification occurs within 1, 2, 3, 4, 5, ormore days after stimulation (and this may depend on the type oftransduction used; for example with retroviral vectors it is within 2days).

The genetic modification of the NKT cells can occur by the hand of man,and in particular embodiments the genetic modification renders the cellsable to specifically target one or more cancer cells, such as cancercells that express a particular antigen, for example. In specificembodiments, the modification provides the NKT cells with a particularnon-natural receptor for a certain cancer antigen. The receptor may beof any kind, but in specific embodiments the receptor is a T-cellreceptor or a chimeric antigen receptor, for example. In some cases, thechimeric antigen receptor is for CD19, CD22, CD30, GD2, GPC3, CSPG4,HER2, CEA, Mesothelin, etc.

T-cell receptor for MHC/peptide complexes from non-mutatedtumor-associated antigens (e.g., Survivin, MYCN, NY-ESO1, MAGE, PRAME,WT1, etc.) or patient-specific mutated tumor antigens revealed bysequencing of tumor DNA.

In some cases, the NKT cells are modified to express a CAR. Geneticengineering of the NKT cells to express tumor-directed chimeric antigenreceptors (CAR) can produce antitumor effector cells that bypass tumorimmune escape mechanisms that are due to abnormalities inprotein-antigen processing and presentation. Moreover, these transgenicreceptors can be directed to tumor-associated antigens that are notprotein-derived. In certain embodiments of the disclosure there are NKTcells that are modified to comprise at least a CAR. In specific aspects,a particular NKT cell comprises expression of two or more CARs.

The present disclosure includes NKT cells that express an artificial Tcell receptor referred to as a CAR (that also may be called chimeric Tcell receptors or chimeric immunoreceptors). In embodiments of thedisclosure it is specific for a cancer antigen. The CAR generally mayinclude an ectodomain, transmembrane domain, and endodomain. It may befirst generation, second generation, or third generation, in specificembodiments.

In particular cases, NKT cells include a CAR that is chimeric,non-natural, engineered at least in part by the hand of man, anddirected to a particular cancer antigen of interest. In particularcases, the engineered CAR has one, two, three, four, or more components,and in some embodiments the one or more components facilitate targetingor binding of the NKT cell to the cancer antigen-comprising cancer cell.In specific embodiments, the CAR comprises an antibody for the cancerantigen, part or all of a cytoplasmic signaling domain, and/or part orall of one or more co-stimulatory molecules, for example endodomains ofco-stimulatory molecules. In specific embodiments, the antibody is asingle-chain variable fragment (scFv). In certain aspects the antibodyis directed at cancer antigens on the cell surface of cancer cells thatexpress an antigen of interest, for example. In certain embodiments, acytoplasmic signaling domain, such as those derived from the T cellreceptor ζ-chain, is employed as at least part of the chimeric receptorin order to produce stimulatory signals for NKT cell proliferation andeffector function following engagement of the chimeric receptor with thetarget antigen. Examples would include, but are not limited to,endodomains from co-stimulatory molecules such as CD27, CD28, 4-1BB, andOX40 or the signaling components of cytokine receptors such as IL7 andIL15. In particular embodiments, co-stimulatory molecules are employedto enhance the activation, proliferation, and cytotoxicity of the NKTcells produced by the CAR after antigen engagement. In specificembodiments, the co-stimulatory molecules are CD28, OX40, and 4-1BB.

In general, an ectodomain of the CAR encompasses a signal peptide,antigen recognition domain, and a spacer that links the antigenrecognition domain to the transmembrane domain. The antigen recognitiondomain generally will comprise a single chain variable fragment (scFv)specific for a particular cancer antigen. However, in cases whereinthere are two or more CARs in the same cell, the second CAR may comprisean scFv specific for another particular antigens. Examples of cancerantigens include any one of Melanoma-associated antigen (MAGE),Preferentially expressed antigen of melanoma (PRAME), CD19, CD20, CD22,κ-light chain, CD30, CD33, CD123, CD38, CD138, ROR1, ErbB2, ErbB3/4,EGFr vIII, carcinoembryonic antigen, EGP2, EGP40, HER2, mesothelin,TAG72, PSMA, NKG2D ligands, B7-H6, IL-13 receptor a2, MUC1, MUC16, CA9,GD2, GD3, HMW-MAA, CD171, Lewis Y, G250/CAIX, HLA-AI MAGE AI, HLA-A2NY-ESO-1, PSC1, folate receptor-a, CD44v6, CD44v7/8, 8H9, NCAM, VEGFreceptors, 5T4, Fetal AchR, NKG2D ligands, or CD44v6, for example.

Examples of hinge regions for the ectodomain include the CH2CH3 regionof immunoglobulin, the hinge region from IgG1, and portions of CD3. Thetransmembrane region may be of any kind, although in some cases it isCD28.

In general, the endodomain of the CAR of the disclosure is utilized forsignal transmission in the cell after antigen recognition and cluster ofthe receptors. The most commonly used endodomain component is CD3-zetathat contains 3 ITAMs and that transmits an activation signal to the Tcell after the antigen is bound. In some embodiments, additionalco-stimulatory signaling is utilized, such as CD3-zeta in combinationwith CD28, 4-1BB, and/or OX40.

IV. Cells Generally

Cells of the disclosure include both CD62L-positive NKT cells that havebeen co-stimulated by one or more costimulatory agents as well as theparticular costimulatory agent that itself is an artificial antigenpresenting cell (which may be referred to as a non-natural cell that hasantigen presenting cell activity). In some embodiments, there is as acomposition of matter, an artificial antigen presenting cell that is anon-natural cell expressing CD1d and that expresses one or more ligandsof one or more costimulatory receptors.

As used herein, the terms “cell,” “cell line,” and “cell culture” may beused interchangeably. All of these terms also include their progeny,which is any and all subsequent generations. It is understood that allprogeny may not be identical due to deliberate or inadvertent mutations.In the context of expressing a heterologous nucleic acid sequence, a“host cell” can refer to a prokaryotic or cukaryotic cell, and itincludes any transformable organism that is capable of replicating avector and/or expressing a heterologous gene encoded by a vector. A hostcell can, and has been, used as a recipient for vectors. A host cell maybe “transfected” or “transformed,” which refers to a process by whichexogenous nucleic acid is transferred or introduced into the host cell.A transformed cell includes the primary subject cell and its progeny. Asused herein, the terms “engineered” and “recombinant” cells or hostcells are intended to refer to a cell into which an exogenous nucleicacid sequence, such as, for example, a vector, has been introduced.Therefore, recombinant cells are distinguishable from naturallyoccurring cells which do not contain a recombinantly introduced nucleicacid.

In certain embodiments, it is contemplated that RNAs or proteinaceoussequences may be co-expressed with other selected RNAs or proteinaceoussequences in the same host cell. Co-expression may be achieved byco-transfecting the host cell with two or more distinct recombinantvectors. Alternatively, a single recombinant vector may be constructedto include multiple distinct coding regions for RNAs, which could thenbe expressed in host cells transfected with the single vector.

Some vectors may employ control sequences that allow it to be replicatedand/or expressed in both prokaryotic and eukaryotic cells. One of skillin the art would further understand the conditions under which toincubate all of the above described host cells to maintain them and topermit replication of a vector. Also understood and known are techniquesand conditions that would allow large-scale production of vectors, aswell as production of the nucleic acids encoded by vectors and theircognate polypeptides, proteins, or peptides.

The cells used in the disclosure are eukaryotic, including mammalian,although prokaryotic cells may be employed for manipulation inrecombinant engineering of vectors or DNA to integrate into the vectors.The cells are particularly human, but can be associated with any animalof interest, particularly domesticated animals, such as equine, bovine,murine, ovine, canine, feline, etc., for use in their respective animal.

The cells can be autologous cells, syngeneic cells, allogenic cells andeven in some cases, xenogeneic cells, such as in relation to theindividual that is receiving the cells. The cells may be modified bychanging the major histocompatibility complex (“MHC”) profile, byinactivating β₂-microglobulin to prevent the formation of functionalClass I MHC molecules, inactivation of Class II molecules, providing forexpression of one or more MHC molecules, enhancing or inactivatingcytotoxic capabilities by enhancing or inhibiting the expression ofgenes associated with the cytotoxic activity, or the like.

Expression vectors that encode a CAR can be introduced into the cells asone or more DNA molecules or constructs, where there may be at least onemarker that will allow for selection of host cells that contain theconstruct(s). The constructs can be prepared in conventional ways, wherethe genes and regulatory regions may be isolated, as appropriate,ligated, cloned in an appropriate cloning host, analyzed by restrictionor sequencing, or other convenient means. Particularly, using PCR,individual fragments including all or portions of a functional unit maybe isolated, where one or more mutations may be introduced using “primerrepair”, ligation, in vitro mutagenesis, etc., as appropriate. Theconstruct(s) once completed and demonstrated to have the appropriatesequences may then be introduced into the CTL by any convenient means.The constructs may be integrated and packaged into non-replicating,defective viral genomes like Adenovirus, Adeno-associated virus (AAV),or Herpes simplex virus (HSV) or others, including retroviral vectors,for infection or transduction into cells. The constructs may includeviral sequences for transfection, if desired. Alternatively, theconstruct may be introduced by fusion, electroporation, biolistics,transfection, lipofection, or the like. The host cells may be grown andexpanded in culture before introduction of the construct(s), followed bythe appropriate treatment for introduction of the construct(s) andintegration of the construct(s). The cells are then expanded andscreened by virtue of a marker present in the construct. Various markersthat may be used successfully include hprt, neomycin resistance,thymidine kinase, hygromycin resistance, etc.

In many situations one may wish to be able to kill the modified cells,where one wishes to terminate the treatment, the cells becomeneoplastic, in research where the absence of the cells after theirpresence is of interest, or other event. For this purpose one canprovide for the expression of certain gene products in which one cankill the modified cells under controlled conditions. Suicide geneproducts, such as caspase 9, are examples of such products.

By way of illustration, cancer patients or patients susceptible tocancer or suspected of having cancer may be treated as follows. Cellsmodified as described herein may be administered to the patient andretained for extended periods of time.

The individual may receive one or more administrations of the cells. Thecell(s) would be modified and provided to the individual in needthereof.

V. Polynucleotides

The present disclosure also encompasses a composition comprising anucleic acid sequence encoding an antigen-specific CAR as defined hereinand cells harboring the nucleic acid sequence. The nucleic acid moleculeis a recombinant nucleic acid molecule, in particular aspects and may besynthetic. It may comprise DNA, RNA as well as PNA (peptide nucleicacid) and it may be a hybrid thereof.

Furthermore, it is envisaged for further purposes that nucleic acidmolecules may contain, for example, thioester bonds and/or nucleotideanalogues. The modifications may be useful for the stabilization of thenucleic acid molecule against endo- and/or exonucleases in the cell. Thenucleic acid molecules may be transcribed by an appropriate vectorcomprising a chimeric gene that allows for the transcription of saidnucleic acid molecule in the cell. In this respect, it is also to beunderstood that such polynucleotides can be used for “gene targeting” or“gene therapeutic” approaches. In another embodiment the nucleic acidmolecules are labeled. Methods for the detection of nucleic acids arewell known in the art, e.g., Southern and Northern blotting, PCR orprimer extension. This embodiment may be useful for screening methodsfor verifying successful introduction of the nucleic acid moleculesdescribed above during gene therapy approaches.

The nucleic acid molecule(s) may be a recombinantly produced chimericnucleic acid molecule comprising any of the aforementioned nucleic acidmolecules either alone or in combination. In specific aspects, thenucleic acid molecule is part of a vector.

The present disclosure therefore also relates to a compositioncomprising a vector comprising the nucleic acid molecule described inthe present disclosure.

Many suitable vectors are known to those skilled in molecular biology,the choice of which would depend on the function desired and includeplasm ids, cosmids, viruses, bacteriophages and other vectors usedconventionally in genetic engineering. Methods that are well known tothose skilled in the art can be used to construct various plasm ids andvectors; see, for example, the techniques described in Sambrook et al.(1989) and Ausubel, Current Protocols in Molecular Biology, GreenPublishing Associates and Wiley Interscience, N.Y. (1989), (1994).Alternatively, the polynucleotides and vectors of the disclosure can bereconstituted into liposomes for delivery to target cells. A cloningvector may be used to isolate individual sequences of DNA. Relevantsequences can be transferred into expression vectors where expression ofa particular polypeptide is required. Typical cloning vectors includepBluescript SK, pGEM, pUC9, pBR322 and pGBT9. Typical expression vectorsinclude pTRE, pCAL-n-EK, pESP-1, pOP13CAT.

In specific embodiments, there is a vector that comprises a nucleic acidsequence that is a regulatory sequence operably linked to the nucleicacid sequence encoding an antigen-specific CAR defined herein. Suchregulatory sequences (control elements) are known to the artisan and mayinclude a promoter, a splice cassette, translation initiation codon,translation and insertion site for introducing an insert into thevector. In specific embodiments, the nucleic acid molecule isoperatively linked to said expression control sequences allowingexpression in eukaryotic or prokaryotic cells.

It is envisaged that a vector is an expression vector comprising thenucleic acid molecule encoding an antigen-specific CAR as definedherein. In specific aspects, the vector is a viral vector, such as alentiviral vector. Lentiviral vectors are commercially available,including from Clontech (Mountain View, Calif.) or GeneCopoeia(Rockville, Md.), for example.

The term “regulatory sequence” refers to DNA sequences that arenecessary to effect the expression of coding sequences to which they areligated. The nature of such control sequences differs depending upon thehost organism. In prokaryotes, control sequences generally includepromoters, ribosomal binding sites, and terminators. In eukaryotesgenerally control sequences include promoters, terminators and, in someinstances, enhancers, transactivators or transcription factors. The term“control sequence” is intended to include, at a minimum, all componentsthe presence of which are necessary for expression, and may also includeadditional advantageous components.

The term “operably linked” refers to a juxtaposition wherein thecomponents so described are in a relationship permitting them tofunction in their intended manner. A control sequence “operably linked”to a coding sequence is ligated in such a way that expression of thecoding sequence is achieved under conditions compatible with the controlsequences. In case the control sequence is a promoter, it is obvious fora skilled person that double-stranded nucleic acid is preferably used.

Thus, the recited vector is an expression vector, in certainembodiments. An “expression vector” is a construct that can be used totransform a selected host and provides for expression of a codingsequence in the selected host. Expression vectors can for instance becloning vectors, binary vectors or integrating vectors. Expressioncomprises transcription of the nucleic acid molecule preferably into atranslatable mRNA. Regulatory elements ensuring expression inprokaryotes and/or eukaryotic cells are well known to those skilled inthe art. In the case of eukaryotic cells they comprise normallypromoters ensuring initiation of transcription and optionally poly-Asignals ensuring termination of transcription and stabilization of thetranscript. Possible regulatory elements permitting expression inprokaryotic host cells comprise, e.g., the P_(L), lac, trp or tacpromoter in E. coli, and examples of regulatory elements permittingexpression in eukaryotic host cells are the AOX1 or GAL1 promoter inyeast or the CMV-, SV40-, RSV-promoter (Rous sarcoma virus),CMV-enhancer, SV40-enhancer or a globin intron in mammalian and otheranimal cells.

Beside elements that are responsible for the initiation of transcriptionsuch regulatory elements may also comprise transcription terminationsignals, such as the SV40-poly-A site or the tk-poly-A site, downstreamof the polynucleotide. Furthermore, depending on the expression systemused leader sequences capable of directing the polypeptide to a cellularcompartment or secreting it into the medium may be added to the codingsequence of the recited nucleic acid sequence and are well known in theart. The leader sequence(s) is (are) assembled in appropriate phase withtranslation, initiation and termination sequences, and preferably, aleader sequence capable of directing secretion of translated protein, ora portion thereof, into the periplasmic space or extracellular medium.Optionally, the heterologous sequence can encode a fusion proteinincluding an N-terminal identification peptide imparting desiredcharacteristics, e.g., stabilization or simplified purification ofexpressed recombinant product; see supra. In this context, suitableexpression vectors are known in the art such as Okayama-Berg cDNAexpression vector pcDV1 (Pharmacia), pEF-Neo, pCDM8, pRc/CMV, pcDNA1,pcDNA3 (Invitrogen), pEF-DHFR and pEF-ADA, (Raum et al. Cancer ImmunolImmunother (2001) 50(3), 141-150) or pSPORT1 (GIBCO BRL).

In some embodiments, the expression control sequences are eukaryoticpromoter systems in vectors capable of transforming of transfectingeukaryotic host cells, but control sequences for prokaryotic hosts mayalso be used. Once the vector has been incorporated into the appropriatehost, the host is maintained under conditions suitable for high levelexpression of the nucleotide sequences, and as desired, the collectionand purification of the polypeptide of the disclosure may follow. Inparticular embodiments, one or more encodable sequences are regulated byexpression control sequences that are responsive to hypoxicenvironments.

Additional regulatory elements may include transcriptional as well astranslational enhancers. Advantageously, the above-described vectors ofthe disclosure comprises a selectable and/or scorable marker. Selectablemarker genes useful for the selection of transformed cells are wellknown to those skilled in the art and comprise, for example,antimetabolite resistance as the basis of selection for dhfr, whichconfers resistance to methotrexate (Reiss, Plant Physiol. (Life-Sci.Adv.) 13 (1994), 143-149); npt, which confers resistance to theaminoglycosides neomycin, kanamycin and paromycin (Herrera-Estrella,EMBO J. 2 (1983), 987-995) and hygro, which confers resistance tohygromycin (Marsh, Gene 32 (1984), 481-485). Additional selectable geneshave been described, namely trpB, which allows cells to utilize indolein place of tryptophan; hisD, which allows cells to utilize histinol inplace of histidine (Hartman, Proc. Natl. Acad. Sci. USA 85 (1988),8047); mannose-6-phosphate isomerase which allows cells to utilizemannose (WO 94/20627) and ODC (ornithine decarboxylase) which confersresistance to the ornithine decarboxylase inhibitor,2-(difluoromethyl)-DL-omithine, DFMO (McConlogue, 1987, In: CurrentCommunications in Molecular Biology, Cold Spring Harbor Laboratory ed.)or deaminase from Aspergillus terreus which confers resistance toBlasticidin S (Tamura, Biosci. Biotechnol. Biochem. 59 (1995),2336-2338).

Useful scorable markers are also known to those skilled in the art andare commercially available. Advantageously, said marker is a geneencoding luciferase (Giacomin, P I. Sci. 116 (1996), 59-72; Scikantha,J. Bact. 178 (1996), 121), green fluorescent protein (Gerdes, FEBS Lett.389 (1996), 44-47) or β-glucuronidase (Jefferson, EMBO J. 6 (1987),39013907). This embodiment is particularly useful for simple and rapidscreening of cells, tissues and organisms containing a recited vector.

As described above, the recited nucleic acid molecule can be used in acell, alone, or as part of a vector to express the encoded polypeptidein cells. The nucleic acid molecules or vectors containing the DNAsequence(s) encoding any one of the specific CAR constructs isintroduced into the cells that in turn produce the polypeptide ofinterest. The recited nucleic acid molecules and vectors may be designedfor direct introduction or for introduction via liposomes, or viralvectors (e.g., adenoviral, retroviral) into a cell.

In accordance with the above, the present disclosure relates to methodsto derive vectors, particularly plasm ids, cosmids, viruses andbacteriophages used conventionally in genetic engineering that comprisea nucleic acid molecule encoding the polypeptide sequence of anantigen-specific CAR defined herein. Preferably, said vector is anexpression vector and/or a gene transfer or targeting vector. Expressionvectors derived from viruses such as retroviruses, vaccinia virus,adeno-associated virus, herpes viruses, or bovine papilloma virus, maybe used for delivery of the recited polynucleotides or vector intotargeted cell populations. Methods which are well known to those skilledin the art can he used to construct recombinant vectors; see, forexample, the techniques described in Sambrook et al. (loc cit.), Ausubel(1989, loc cit.) or other standard text books. Alternatively, therecited nucleic acid molecules and vectors can be reconstituted intoliposomes for delivery to target cells. The vectors containing thenucleic acid molecules of the disclosure can be transferred into thehost cell by well-known methods, which vary depending on the type ofcellular host. For example, calcium chloride transfection is commonlyutilized for prokaryotic cells, whereas calcium phosphate treatment orelectroporation may he used for other cellular hosts; see Sambrook,supra.

XII. Pharmaceutical Compositions

In accordance with this disclosure, the term “pharmaceuticalcomposition” relates to a composition for administration to anindividual. In specific aspects of the disclosure, the pharmaceuticalcomposition comprises a plurality of NKT cells. In a preferredembodiment, the pharmaceutical composition comprises a composition forparenteral, transdermal, intraluminal, intra-arterial, intrathecal orintravenous administration or for direct injection into a cancer. It isin particular envisaged that said pharmaceutical composition isadministered to the individual via infusion or injection. Administrationof the suitable compositions may be effected by different ways, e.g., byintravenous, subcutaneous, intraperitoneal, intramuscular, topical orintradermal administration.

The pharmaceutical composition of the present disclosure may furthercomprise a pharmaceutically acceptable carrier. Examples of suitablepharmaceutical carriers are well known in the art and include phosphatebuffered saline solutions, water, emulsions, such as oil/wateremulsions, various types of wetting agents, sterile solutions, etc.Compositions comprising such carriers can be formulated by well-knownconventional methods. These pharmaceutical compositions can beadministered to the subject at a suitable dose.

The dosage regimen will be determined by the attending physician andclinical factors. As is well known in the medical arts, dosages for anyone patient depends upon many factors, including the patient's size,body surface area, age, the particular compound to be administered, sex,time and route of administration, general health, and other drugs beingadministered concurrently. Progress can be monitored by periodicassessment. CAR-modified NKT may be administered via intravenousinfusion. Doses can range from 1×10⁷/m² to 2×10⁸/m², and in specificembodiments up to 10⁹ cells may be utilized.

The compositions of the disclosure may be administered locally orsystemically. Administration will generally be parenteral, e.g.,intravenous; DNA may also be administered directly to the target site,e.g., by biolistic delivery to an internal or external target site or bycatheter to a site in an artery. In a preferred embodiment, thepharmaceutical composition is administered subcutaneously and in an evenmore preferred embodiment intravenously. Preparations for parenteraladministration include sterile aqueous or non-aqueous solutions,suspensions, and emulsions. Examples of non-aqueous solvents arepropylene glycol, polyethylene glycol, vegetable oils such as olive oil,and injectable organic esters such as ethyl oleate. Aqueous carriersinclude water, alcoholic/aqueous solutions, emulsions or suspensions,including saline and buffered media. Parenteral vehicles include sodiumchloride solution, Ringer's dextrose, dextrose and sodium chloride,lactated Ringer's, or fixed oils. Intravenous vehicles include fluid andnutrient replenishes, electrolyte replenishers (such as those based onRinger's dextrose), and the like. Preservatives and other additives mayalso be present such as, for example, antimicrobials, anti-oxidants,chelating agents, and inert gases and the like. In addition, thepharmaceutical composition of the present disclosure might compriseproteinaceous carriers, like, e.g., serum albumin or immunoglobulin,preferably of human origin. It is envisaged that the pharmaceuticalcomposition of the disclosure might comprise, in addition to the CARconstructs or nucleic acid molecules or vectors encoding the same (asdescribed in this disclosure), further biologically active agents,depending on the intended use of the pharmaceutical composition.

XII. Therapeutic Uses of NKT Cells

By way of illustration, cancer patients or patients susceptible tocancer or suspected of having cancer may be treated as described herein.NKT cells modified as described herein may be administered to theindividual and retained for extended periods of time. The individual mayreceive one or more administrations of the cells. In some embodiments,the genetically modified cells are encapsulated to inhibit immunerecognition and placed at the site of the tumor.

In various embodiments the expression constructs, nucleic acidsequences, vectors, host cells and/or pharmaceutical compositionscomprising the same are used for the prevention, treatment oramelioration of a cancerous disease, such as a tumorous disease. Inparticular embodiments, the pharmaceutical composition of the presentdisclosure may be particularly useful in preventing, ameliorating and/ortreating cancer, including cancer having solid tumors, for example.

As used herein “treatment” or “treating,” includes any beneficial ordesirable effect on the symptoms or pathology of a disease orpathological condition, and may include even minimal reductions in oneor more measurable markers of the disease or condition being treated,e.g., cancer. Treatment can involve optionally either the reduction oramelioration of symptoms of the disease or condition, or the delaying ofthe progression of the disease or condition. “Treatment” does notnecessarily indicate complete eradication or cure of the disease orcondition, or associated symptoms thereof.

As used herein, “prevent,” and similar words such as “prevented,”“preventing” etc., indicate an approach for preventing, inhibiting, orreducing the likelihood of the occurrence or recurrence of, a disease orcondition, e.g., cancer. It also refers to delaying the onset orrecurrence of a disease or condition or delaying the occurrence orrecurrence of the symptoms of a disease or condition. As used herein,“prevention” and similar words also includes reducing the intensity,effect, symptoms and/or burden of a disease or condition prior to onsetor recurrence of the disease or condition.

In particular embodiments, the present disclosure contemplates, in part,cells harboring expression constructs, nucleic acid molecules and/orvectors that can administered either alone or in any combination withanother therapy, and in at least some aspects, together with apharmaceutically acceptable carrier or excipient. In certainembodiments, prior to administration of the cells, said nucleic acidmolecules or vectors may be stably integrated into the genome of thecells. In specific embodiments, viral vectors may be used that arespecific for certain cells or tissues and persist in said cells.Suitable pharmaceutical carriers and excipients are well known in theart. The compositions prepared according to the disclosure can be usedfor the prevention or treatment or delaying the above identifieddiseases.

Furthermore, the disclosure relates to a method for the prevention,treatment or amelioration of a cancerous (including tumorous) diseasecomprising the step of administering to a subject in need thereof aneffective amount of cells harboring an antigen recognition moietymolecule and a chemotherapy resistance molecule, nucleic acid sequencethat encodes them, vector(s) that encodes them, as contemplated hereinand/or produced by a process as contemplated herein.

Possible indications for administration of the composition(s) of theexemplary modified immune cells are cancerous diseases, includingtumorous diseases, including breast, prostate, lung, and colon cancersor epithelial cancers/carcinomas such as MM, breast cancer, coloncancer, prostate cancer, head and neck cancer, skin cancer, cancers ofthe genito-urinary tract, e.g., ovarian cancer, endometrial cancer,cervix cancer and kidney cancer, lung cancer, gastric cancer, cancer ofthe small intestine, liver cancer, pancreas cancer, gall bladder cancer,cancers of the bile duct, esophagus cancer, cancer of the salivaryglands and cancer of the thyroid gland, neuroblastoma, medulloblastoma,glioblastoma, hematopoetic malignancies, and so forth. Exemplaryindications for administration of the composition(s) of cells arecancerous diseases, including any malignancies that express a particularantigen, for example. In addition, it includes malignancies thataberrantly express other tumor antigens and those may also be targeted.The administration of the composition(s) of the disclosure is useful forall stages and types of cancer, including for minimal residual disease,early cancer, advanced cancer, and/or metastatic cancer and/orrefractory cancer, for example.

The disclosure further encompasses co-administration protocols withother compounds, e.g. bispecific antibody constructs, targeted toxins orother compounds, which act via immune cells. The clinical regimen forco-administration of the inventive compound(s) may encompassco-administration at the same time, before and/or after theadministration of the other component. Particular combination therapiesinclude chemotherapy, radiation, surgery, hormone therapy, or othertypes of immunotherapy.

Embodiments relate to a kit comprising one or more NKT cells asdescribed herein, a nucleic acid sequence as described herein, a vectoras described herein and/or a host as described herein. It is alsocontemplated that the kit of this disclosure comprises a pharmaceuticalcomposition as described herein above, either alone or in combinationwith further medicaments to be administered to an individual in need ofmedical treatment or intervention.

The NKT cells that have been modified with the construct(s) are thengrown in culture under selective conditions and cells that are selectedas having the construct may then be expanded and further analyzed,using, for example; the polymerase chain reaction for determining thepresence of the construct(s) in the host cells. Once the modified hostcells have been identified, they may then be used as planned, e.g.,expanded in culture or introduced into a host organism.

Depending upon the nature of the cells, the cells may be introduced intoa host organism, e.g., a mammal, in a wide variety of ways. The cellsmay be introduced at the site of the tumor, in specific embodiments,although in alternative embodiments the cells hone to the cancer or aremodified to hone to the cancer. The number of cells that are employedwill depend upon a number of circumstances, the purpose for theintroduction, the lifetime of the cells, the protocol to be used, forexample, the number of administrations, the ability of the cells tomultiply, the stability of the recombinant construct, and the like. Thecells may be applied as a dispersion, generally being injected at ornear the site of interest. The cells may be in aphysiologically-acceptable medium.

The DNA introduction need not result in integration in every case. Insome situations, transient maintenance of the DNA introduced may besufficient. In this way, one could have a short term effect, where cellscould be introduced into the host and then turned on after apredetermined time, for example, after the cells have been able to hometo a particular site.

The cells may be administered as desired. Depending upon the responsedesired, the manner of administration, the life of the cells, the numberof cells present, various protocols may be employed. The number ofadministrations will depend upon the factors described above at least inpart.

It should be appreciated that the system is subject to many variables,such as the cellular response to the ligand, the efficiency ofexpression and, as appropriate, the level of secretion, the activity ofthe expression product, the particular need of the patient, which mayvary with time and circumstances, the rate of loss of the cellularactivity as a result of loss of cells or expression activity ofindividual cells, and the like. Therefore, it is expected that for eachindividual patient, even if there were universal cells which could beadministered to the population at large, each patient would be monitoredfor the proper dosage for the individual, and such practices ofmonitoring a patient are routine in the art.

VI. Kits of the Disclosure

Any of the compositions described herein may be comprised in a kit. In anon-limiting example, cells or reagents to manipulate cells may becomprised in a kit. In certain embodiments, NKT cells or a population ofcells that comprises NKT cells may be comprised in a kit. Such a kit mayor may not have one or more reagents for manipulation of cells. Suchreagents include small molecules, proteins, nucleic acids, antibodies,buffers, primers, nucleotides, salts, and/or a combination thereof, forexample. Nucleotides that encode one or more cytokines, or cytokinesthemselves, may he included in the kit. Proteins, such as cytokines orantibodies, including agonistic monoclonal antibodies, may be includedin the kit. Substrates that comprise the antibodies, or naked substratesthemselves, may be included in the kit, and in some embodiments reagentsto generate antibody-bearing substrates are included in the kit. Thesubstrates may be of any kind including a bead or plate. Cells thatcomprise antigen presenting cell activity or reagents to generate samemay be included in the kit. Nucleotides that encode chimeric antigenreceptors or T-cell receptors may be included in the kit, includingreagents to generate same.

In particular aspects, the kit comprises the cell therapy of thedisclosure and also another cancer therapy. In some cases, the kit, inaddition to the cell therapy embodiments, also includes a second cancertherapy, such as chemotherapy, hormone therapy, and/or immunotherapy,for example. The kit(s) may be tailored to a particular cancer for anindividual and comprise respective second cancer therapies for theindividual.

The kits may comprise suitably aliquoted compositions of the presentinvention. The components of the kits may be packaged either in aqueousmedia or in lyophilized form. The container means of the kits willgenerally include at least one vial, test tube, flask, bottle, syringeor other container means, into which a component may be placed, andpreferably, suitably aliquoted. Where there are more than one componentin the kit, the kit also may generally contain a second, third or otheradditional container into which the additional components may beseparately placed. However, various combinations of components may becomprised in a vial. The kits of the present invention also willtypically include a means for containing the composition and any otherreagent containers in close confinement for commercial sale. Suchcontainers may include injection or blow-molded plastic containers intowhich the desired vials are retained.

When the components of the kit are provided in one and/or more liquidsolutions, the liquid solution is an aqueous solution, with a sterileaqueous solution being particularly preferred. In which case, thecontainer means may itself be a syringe, pipette, and/or other such likeapparatus, from which the formulation may be applied to an infected areaof the body, injected into an animal, and/or even applied to and/ormixed with the other components of the kit. However, the components ofthe kit may be provided as dried powder(s). When reagents and/orcomponents are provided as a dry powder, the powder can be reconstitutedby the addition of a suitable solvent. It is envisioned that the solventmay also be provided in another container means.

EXAMPLES

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventor to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

Example 1—The Identification of the NKT-Cell Subset Responsible for InVivo Persistence and Therapeutic Activity and Defining the ConditionsRequired for Propagation if This Subset in Culture

The adoptive transfer of invariant Natural Killer T cells (NKTs) isbeing developed as a promising therapeutic modality for immunotherapy ofcancer, autoimmunity, and other diseases. Because NKT-cell frequency islow in human peripheral blood, such therapies require extensive ex vivoexpansion of primary NKTs while preserving their longevity and function.However, cellular and molecular mechanisms responsible for themaintenance of NKTs either in vivo or ex vivo remain largely unknown.Here it is demonstrated that antigen-induced in vitro expansion ofprimary human NKTs is associated with the progressive accumulation ofCD62L-positive subset in all 5 examined individuals regardless of theinitial frequency of that subset. Following magnetic sorting of NKTsinto CD62L-positive and CD62L-negative subsets, only CD62L-positivecells survived and proliferated in response to TCR-stimulation whereasabout 90% of CD62L-negative cells underwent apoptosis within 3 days.Moreover, CD62L-positive NKTs persisted 5 times longer thanCD62L-negative ones after adoptive transfer to NSG mice. Importantly,CD62L-positive NKTs had much higher therapeutic activity andsignificantly prolonged survival of mice in a xenogenic lymphoma model.Proliferating CD62L-positive cells downregulated or maintained CD62Lexpression when they were activated via TCR alone or with co-stimulatoryreceptors, respectively. In particular, certain combinations ofagonistic mAbs for CD3, CD28, and/or 4-1BB enable stable CD62Lexpression on in vitro stimulated NKTs that is associated with themaximal rate of their expansion and subsequent in vivo persistence.Therefore, the results reveal previously unanticipated functionalhierarchy in human NKTs that can be exploited for their effective exvivo expansion for cell therapy applications.

Thus, identified herein is CD62L as a marker of human NKT cells withhigh proliferative potential and superior therapeutic activity. In vitrostimulation conditions were determined that prevent CD62L downregulationon NKTs during ex vivo expansion that is useful for the generation ofNKT-cell products with high therapeutic activity.

Example 2—CD62L+ NKT Cells Have Superior In Vivo Persistence andAnti-Tumor Activity

CD62L+ cells accumulate in culture upon antigenic stimulation of primaryNKTs. Previous studies that compared the phenotype of human NKTs inadult peripheral blood with that in cord blood observed much higherproportions of CD4+ and CD62L+ NKTs in neonates (Baev et al., 2004;D'Andrea et al., 2000; Eger et al., 2006) (FIG. 5A). The prevalence ofCD4+ CD62L+ NKTs in cord blood suggests that the expression of CD4or/and CD62L marks a subset of NKTs that has superior developmentalpotential and could support ex vivo expansion of NKTs for therapeuticapplications. To test this embodiment, immunophenotyping was performedof primary NKTs immediately after isolation from peripheral blood and atdifferent time intervals in culture after stimulation with αGalCer,which consistently produced higher frequency and absolute numbers ofNKTs compared to the use of a T-cell expansion protocol based onCD3/CD28 stimulation (FIG. 6). Despite a notable inter-individualvariability of CD62L expression in freshly isolated NKTs, there was astriking accumulation of CD62L+ fraction of NKTs from 33.63%±27.62% infreshly isolated NKTs to 69.92%±10.57% on day 12 of the culture(P<0.001, FIGS. 1A, 1B). Although CD62L was more frequently expressed onCD4+ NKTs both before and after culture, the accumulation of CD62L+cells could not be explained by a preferential expansion of CD4+ NKTs.Indeed, at the end of a 12-day culture, the frequency of CD62L+, CD4+,and CD62L+ CD4+ NKTs increased 3.9±1.8, 1.9±0.7, and 3.8±1.9 fold,respectively. Consistent with these results, there was an enrichment ofthe CD62L+ CD4-subset at day 12 compared to day 0 (FIG. 5B). Furthermulti-parameter characterization of NKTs showed that CD62L was oftenco-expressed with CCR7 before culture, but CCR7 expression wasprogressively lost during culture (FIG. 5C). Nearly all NKTs expressedboth CD27 and CD28 before culture. While CD27 was down-regulated inabout half of NKTs by day 12 of culture irrespective to CD62L status,CD28 expression remained intact (FIG. 5C).

CD62L− cells expressed higher levels of CD161 and CD56 (NK-likedifferentiation), but a lower level of IL-7Rα (FIG. 5D). While freshlyisolated NKTs rarely expressed exhaustion markers (PD-1, LAG-3, or 6TIM-3) in either the CD62L+ or CD62L− subsets, the CD62L− subsetpreferentially expressed PD-1 and TIM-3 at day 12 of NKT-cell culture(P=0.0043, 0.0184, FIG. 5C). Moreover, immune-related gene expressionanalysis (nCounter® platform) of CD62L+ and CD62L− NKTs sorted on day 12revealed mRNA up-regulation of genes associated with T-cellsurvival/memory (e.g., LEF1, S1PR1, IL-7Rα, IL21R) in CD62L+ NKTs andwith exhaustion/terminal differentiation in CD62L− NKTs (e.g., PD-1,LAG-3, TIM-3, CD244, CD161, CD56, FIG. 1D). The transcription factorlymphoid enhancer factor 1 (LEF1) was the top immune-related geneoverexpressed in CD62L+ compared to CD62L− NKTs. Intracellular flowcytometry analysis demonstrated that CD62L+ NKTs uniformly expressedLEF1, whereas a major fraction of CD62L− cells was LEF1-negative. SinceLEF1 was recently shown to mediate expansion of murine NKT cellprecursors in part via transcriptional activation of GATA3 geneexpression (Carr et al., 2015), the level of GATA3 protein was analyzedin human NKTs in relation to LEF1 and CD62L levels. GATA3 expressionstrongly correlated with the expression of LEF1 and CD62L in human NKTs(FIG. 5E). Of interest, CD62L+ and CD62L− NKTs expressed the same levelof PLZF, a transcriptional master regulator of NKT-cell functionaldifferentiation (Cohen et al., 2013). Thus, the CD62L+ subsetpredominantly accumulates in culture upon antigenic stimulation ofprimary NKTs and loss of CD62L expression is associated with NK-liketerminal differentiation, exhaustion, and down-regulation ofproproliferative transcriptional regulators.

CD62L+ NKTs are Th-0-like cells capable of numeric expansion. Nαext,NKTs were magnetically sorted from primary culture into CD62L+ andCD62L− subsets and examined their functional properties. FIG. 2Ademonstrates that both subsets were either equally cytotoxic (three ofsix donors) or CD62L− NKTs were more cytotoxic than CD62L+ NKTs (threeof six donors) against CD1d+ DAOY medulloblastoma cells when the targetcells were pulsed with αGalCer. The analysis of cytokine production inαGalCer-stimulated NKTs revealed much higher levels of both IFN-γ andIL-4 production by CD62L+ compared to CD62L− subsets (P<0.001, FIG. 2B).CD62L+ cells exhibited a Th-0-like polarization (a balanced productionof IFN-γ and IL-4, typical of whole population of peripheral blood NKTs)whereas the polarization profile of CD62L− cells could not be determinedunambiguously because of the low absolute amounts of each cytokine.Despite strong up-regulation of IL-23R mRNA expression in the CD62L−subset as determined by the nCounter analysis (FIG. 1D, a potential Th17polarization), neither IL-23R protein expression on the cell surface ofNKTs by FACS nor production of IL-17 upon TCR stimulation by ELISA weredetected.

To examine whether the accumulation of CD62L+ NKTs during in vitroexpansion is because of their preferential survival or proliferation inresponse to antigenic stimulation, their rate of cell death andproliferation was measured after stimulation of the sorted cells withantigen-presenting cells that had been pulsed with αGalCer. On day 3after stimulation, 31%±21% and 74%±7.5% of CD62L+ and CD62L− NKTsunderwent apoptosis, respectively (FIG. 2C). There was a much greaterrate of proliferation in CD62L+ vs. CD62L− subsets as measured by CFSEdilution on day 6 (FIG. 2D). Moreover, the majority of cells thatsurvived and proliferated in the CD62L− group expressed CD62L,suggesting that these cells were progenies of a small subset of CD62L+cells in the original CD62L− fraction. Consistent with these results,there was a striking difference in the numbers of NKTs generated after a6-day culture of sorted CD62L+ vs. CD62L− NKTs with IL-2 alone or withTCRstimulation. Indeed, FIG. 2E (upper panel) demonstrates that CD62L+cells underwent 2.5 and 8 fold numeric expansion with IL-2 alone or withTCR-stimulation, respectively. In contrast, CD62L− NKTs failed to expandin either condition. Although the degree of NKT-cell proliferation inresponse to antigenic stimulation varied from donor to donor, the CD62L−subset contributed little or nothing to NKT-cell expansion in all fivetested donors (FIG. 2E, lower panel). Therefore, CD62L+ NKTs survive andproliferate in response to antigenic stimulation and are responsible forNKT-cell numeric expansion in culture.

CD62L+ subset is responsible for NKTs in vivo persistence andtherapeutic activity. To determine the role of the CD62L+ subset in thein vivo persistence of adoptively transferred NKTs, NKTs were transducedwith firefly luciferase and they were magnetically sorted into CD62L+and CD62L− subsets. The inventors then adoptively transferred the sortedcells to NSG mice. Longitudinal bioluminescent imaging demonstrated thatthe signal from CD62L− cells could be detected until day 2, whereasCD62L+ cells remained detectable up to day 10 (P<0.001, FIGS. 3A, B).Next, the in vivo therapeutic potential of CD62L+ and CD62L− NKT-cellsubsets were compared in a model of CAR-redirected immunotherapy forlymphoma. NKTs were transduced with a CD19-specific CAR containing 4-1BBco-stimulatory endodomain (CAR.CD19, FIG. 7) followed by sorting intoCD62L+ and CD62L− subsets. NOD/SCID/IL2Rγ(null) (NSG) mice were i.v.injected with luciferase-transduced CD19+ Daudi lymphoma cells and fourdays later were divided into two groups to receive CD62L+ or CD62L−CAR.CD19 NKT cells. Both CD62L+ and CD62L− CAR.CD19 NKTs prolonged thesurvival of treated animals compared with untreated control (P<0.001).Importantly, only CD62L+ CAR-NKTs induced sustained tumor regressionwith 7 of 9 treated animals alive, 5 of which were tumor-free, for atleast three months. In contrast, all 10 mice treated with CD62L−CAR-NKTs succumbed to tumor progression (P<0.001, FIGS. 3C, D). Thus,CD62L+ NKTs have extended in vivo persistence and superior therapeuticpotential compared with CD62L− NKTs.

Co-stimulation maintains CD62L+ NKTs and prevents exhaustion. There isgrowing evidence that costimulation plays a role in the activation,survival, and expansion of NKTs (van den Heuvel et al., 2011). Whileresting NKTs express CD28, (FIG. 5C), they express little or no lateco-stimulatory receptors such as 4-1BB and OX40 (FIG. 8A). However,stimulation of NKTs with αGalCer-pulsed autologous PBMC resulted inrapid induction of 4-1BB in all NKTs and OX40 in CD4+ NKTs (FIG. 8A).Because CD62L is transiently down-regulated within the first 24-48 hafter TCR-stimulation (data not shown), the kinetics of 4-1BB and OX40expression were analyzed in CD62L+ and CD62L− NKTs that were sortedprior to stimulation. It was found that 71.13%±18.66% and 51.98%±18.83%of CD62L+ and CD62L− NKTs up-regulated OX40 within 72 h afterstimulation (P=0.0072, FIG. 4A). Similarly, stimulated CD62L+ NKTsexpressed a higher level of 4-1BB compared to CD62L− NKTs (P=0.011, FIG.4A). OX40 was preferentially up-regulated in CD4+ subset of eitherCD62L+ or CD62L− NKTs whereas 4-1BB was up-regulated in all NKTs (FIG.8B).

Next, it was considered whether co-stimulation could counteractexhaustion of in vitro expanded NKTs. CD62L+ sorted NKTs were stimulatedon plates coated with an anti-CD3 agonistic mAb OKT3 alone or incombination with mAbs for CD28, 4-1BB, or both. OX40 was not tested inthese settings because the inventors could not obtain an anti-OX40 mAbwith an agonistic activity. First, co-stimulation with either anti-CD28,anti-4-1BB mAb or both increased the number of NKTs generated in culturewithin 7 days compared with stimulation with anti-CD3 alone at 20 ng/ml(P<0.001, FIG. 4B). In the absence of co-stimulation, increasing theconcentration of OKT3 from 20 ng/ml to 1 μg/ml did not affect the numberof NKTs generated, while co-stimulation was effective in increasingNKT-cell number only when it was combined with the lower concentrationof OKT3 (FIG. 9). Importantly, on day 7 after stimulation with OKT3alone less than half the NKTs were positive for CD62L. The addition ofanti-CD28, anti-4-1BB, or anti-CD28 with anti-4-1BB mAbs resulted in theretention of CD62L expression on 58%±7.1% (P=0.026), 73%±9.2%(P=0.0036), and 73%±6.1% (P=0.0002) of NKTs, respectively (FIG. 4C).Coordinately with the retention of CD62L expression, NKTs provided withcostimulatory signals expressed significantly less PD-1 (P<0.05, FIG.4D). Therefore, engagement of costimulatory receptors during antigenicstimulation supports CD62L expression in proliferating NKTs and preventstheir exhaustion.

Significance of Certain Embodiments of the Disclosure

A critical gap in knowledge of human NKT-cell biology has slowed downthe development of effective NKT cell-based cancer immunotherapy.NKT-cell numeric ex vivo expansion and subsequent in vivopersistence—essential requirements for effective NKT-cell based cell andgene therapy applications—depend on the CD62L+ subset of peripheralblood NKTs. Only CD62L+ NKTs survive and proliferate in response torepeated TCR-stimulation, while CD62L− cells undergo early exhaustionand cell death. Although continuous stimulation of NKTs is associatedwith the loss of CD62L expression, activation of co-stimulatoryreceptors during TCR-stimulation can counteract this process.Specifically, the inventors experimentally determined a uniquecombination of CD86, 4-1BBL and OX40L molecules and the levels of theirco-expression on the surface of an aAPC that enable highly efficientclinical-scale NKT-cell expansion with maximal preservation of CD62Lexpression. In certain embodiments, CAR-NKTs generated using aAPC suchas are encompassed by this disclosure exhibit extended in vivopersistence and superior therapeutic activity against in vivo models oflymphoma and neuroblastoma (as examples of cancer types).

The CD62L+ subset is responsible for the numeric expansion of NKTs uponantigenic stimulation ex vivo. The following findings support the aboveconclusion: i) the fraction of CD62L+ cells is dramatically increasedafter primary NKT-cell stimulation, ii) the majority of sorted CD62L−cells undergo apoptosis, whereas sorted CD62L+ cells proliferate inresponse to identical stimulation; iii) CD62L− NKTs exhibit signs ofterminal differentiation (CD161 and CD56 expression) in freshly isolatedPBMC and rapidly acquire an exhaustion phenotype upon in vitrostimulation as evidenced by marked up-regulation of PD-1 and TIM-3expression and diminished ability to produce cytokines. When similarcharacteristics are observed in T-cell therapeutic products, they areassociated with a subsequent lack of persistence or of objectiveresponses after adoptive transfer to cancer patients (Gattinoni et al.,2005; Klebanoff et al., 2005).

Proliferating NKTs eventually down-regulate CD62L expression and acquirean exhaustion phenotype during in vitro culture. This observation likelyreflects the ontogeny of human peripheral NKTs, as the frequency ofCD62L+ NKTs is lower in adult peripheral blood compared with cord blood(Eger et al., 2006; Der Vliet et al., 2000). In one report, cord bloodNKTs were found not to express CD62L (D'Andrea et al., 2000). The reasonfor the discrepancy between that report and others and with the presentresults is technical, in certain embodiments. M. Constantinides et al.identified a very rare naïve-like population of CD1d-restricted T cellswith a high level of CD62L in peripheral blood (Constantinides et al.,2011). However, these cells do not express the invariant Vα24 chain andhave a lower level of PLZF expression compared with the classical NKTs.Both CD62L+ and CD62L− NKTs in this study expressed equally high levelsof PLZF.

CD62L may represent a common marker of cells responsible for thelong-term maintenance of peripheral NKT and T cells. P. Graef et al.demonstrated that CD62L+ central memory T cells possess stem cellproperties; they could propagate themselves while giving rise toeffector-memory and effector T cells (Graef et al., 2014). In the mostrecently published study, D. Sommermeyer et al. demonstrated that humanCAR.CD19 expressing CD8 or CD4 T cells generated from naïve and centralmemory subsets were more effective against Raji lymphoma xenograftscompared to those generated from effector-memory subsets (Sommermeyer etal., 2015). The authors also found that combining the most potent CD4+and CD8+ CAR-expressing subsets produced synergistic antitumor activityin vivo. Activation of NKTs has been shown to lead to transactivation ofNK and CD8 T cells in murine models and in human clinical trials(Dhodapkar et al., 2009; Vivier et al., 2012), so that the combinationof CAR NKTs with other defined subsets of CAR-expressing lymphocytes maybe a useful therapeutic strategy.

LEF1 and IL-7Rα were among the immune-related genes that were mostoverexpressed in CD62L+ compared to CD62L− NKTs. In a recent report,Carr et al. demonstrated a unique function of LEF1 in the expansion ofmurine Vα14-invariant (iNKT) cells during stage-2 of their thymicdevelopment via direct transcriptional activation of CD127 and c-mycgene expression (Carr et al., 2015). Consistent with the observation ofthe coordinated expression of LEF1 and GATA3 in human NKTs, theseinvestigators also found that LEF1 up-regulates transcription factorGATA3, which is required for IL-4 production in iNKT2 cells as well asfor dual production of IL-4 and INFγ in iNKT1 cells. The latter cellsclosely resemble human peripheral blood NKTs, in which it was found thatCD62L+ cells preferentially express GATA3 and produce high levels ofboth cytokines. Taking the Carr et al. study with murine NKTs and theresults with human NKTs, in specific embodiments LEF1 plays a criticalrole at early stages of NKT-cell development and controls their numberand function. The high level of LEF1 expression in CD62L+ and its lossin CD62L− subsets of human NKTs is consistent with a model of linearprogression from a less differentiated CD62L+ NKTs with a preservedproliferative potential and Th-O-like cytokine profile towardsterminally differentiated CD62L− NKT cells with diminished ability toproliferate and produce cytokines.

There is growing evidence that co-stimulation plays a critical role inthe development, activation, and functional responses of NKTs in murinemodels (van den Heuvel et al., 2011; Uldrich et al., 2005). However,little is known about the expression of co-stimulatory receptors inhuman NKTs. In this disclosure, the inventors focused on a set ofco-stimulatory receptors that have pronounced pro-survival properties inhuman T cells: CD28, 4-1BB, and OX40 (Acuto et al., 2003; Kroczek etal., 2004; Redmond et al., 2009). First, it was confirmed that freshlyisolated human NKTs express CD28 (34) and it was shown that theyco-express CD27, thereby resembling the corresponding stage of memory Tcell differentiation with preserved functional potential (Okada et al.,2008). The disclosure is the first to characterize the baseline andpost-stimulation kinetics of 4-1BB and OX40 in human NKTs. Bothreceptors are undetectable in freshly isolated NKTs but are inducedfollowing TCR-stimulation. Similar to T cells (Croft, 2010), human NKTspreferentially up-regulated OX40 in the CD4+ subset. The majority ofNKTs also up-regulated 4-1BB, which is preferentially up-regulated inthe CD8+ subset of T cells (Lynch, 2008). Importantly, co-stimulation ofindividual co-stimulatory receptors could inhibit the loss of CD62Lexpression and rescue NKTs from exhaustion. There was an additive effecton the maintenance of CD62L+ NKTs when CD28 and 4-1BB weresimultaneously activated.

Example 3—Examples of Methods and Materials

Cell lines and culture conditions. Daudi, Raji, DAOY, Ramos and 293Tcells were purchased from ATCC. Daudi, Raji, and Ramos cells werecultured in RPMI, whereas DAOY and 293T cells were maintained in IMDM(Invitrogen). Both types of medium were supplemented with 10% FBS(Hyclone), 2 mM GlutaMAX-1 (Gibco-BRL).

NKT-cell isolation, transduction, expansion and sorting. To analyze thecord blood NKT cells discarded cord blood units obtained from MDAnderson Cancer Center Cord Blood Bank were used according to theprotocols approved by the Institutional Review Boards at MD AndersonCancer Center and Baylor College of Medicine. PBMC of healthy donors (atleast 18 years old) were isolated by gradient centrifugation from buffycoats purchased from Gulf Coast Regional Blood Center. NKTs werepurified by anti-iNKT microbeads (Miltenyi Biotec). The negative PBMCfraction was irradiated (40 Gy) and aliquoted. NKTs were stimulated withan aliquot of autologous PBMCs pulsed with 100 ng/mL αGalCer (KyowaHakko Kirin). Recombinant IL-2 (200 U/ml, National Cancer InstituteFrederick) was added every other day in complete RPMI (HyClone RPMI1640, 10% heat inactivated fetal bovine serum and 2 mM Glutamax). NKTswere expanded for 10 days and then re-stimulated with autologous PBMC(irradiated with 40 Gy) or Ramos cells as aAPC (irradiated with 100 Gy)when indicated. On day 3 after re-stimulation, 24 well, non-tissueculture plates were coated with retronectin (Takara Bio) and afterwashing inoculated with 1 ml of retroviral supernatant containingCAR.CD19 and spun for 60 min at 4600 G. The viral supernatant was thenremoved and stimulated NKTs were added to the wells in complete mediaand 200 U/ml rhIL-2. Cells were removed from the plate after 48 h,washed, re-suspended at the concentration 10⁶ cell/ml in complete RPMIwith 200 U/ml IL-2 and plated for continued expansion. NKT-cell numberwas determined by Trypan Blue (Life technologies) counting. Whenindicated, NKTs or CAR-NKTs were labeled with CD62L-PE mAb (GREG-56, BDBiosciences) and anti-PE microbeads (Miltenyi) followed by magneticsorting into CD62L+ and CD62L− subsets according to the manufacturer'sinstructions. The phenotype of the sorted cells was determined by FACS.

Retroviral constructs and retrovirus production. CAR.CD19 and CAR.GD2constructs was made as previously described (Heczey et al., 2014; Puleet al., 2005) and contained a scFv from the CD19-specific antibodyFMC-63 or the GD2-specific antibody 14G2a connected via a short spacerderived from the IgG1 hinge region to the transmembrane domain derivedfrom CD8α, followed by signaling endodomain sequences of 4-1BB fusedwith ζ chain. Retroviral supernatants were produced by transfection of293T cells with a combination of chimeric antigen containing plasm ids,RDF plasmid encoding the RD114 envelope and PegPam3 plasmid encoding theMoMLV gag-pol as previously described (Vera et al., 2006).

Proliferation and apoptosis assays. NKTs were labeled with CFSE(Invitrogen) and stimulated with αGalCer-pulsed PBMC, or plates coatedwith 20 ng/ml anti-CD3 (OKT3) alone or in combination with 0.5 ug/mlanti-CD28 (CD28.2), and/or 1.5 ug/ml anti-4-1BB (h41BB-M127) (BDBiosciences). Cell proliferation was examined on day 3 and 6 bymeasuring CFSE dilution using flow cytometry. In addition, early andlate stages of apoptosis were measured on day 3 by staining withAnnexin-V and 7-AAD (BD Biosciences) followed by flow cytometry.

Multiplex cytokine quantification assay. NKTs were stimulated for 24hours with either APCs or agonistic antibody-coated plates (clone 6611,BD Biosciences). Supernatants were collected and analyzed with HumanCytokine/Chemokine Immunoassay kit (Millipore) using the Luminex® assayaccording to the manufacturer's instructions.

Flow cytometry. Immunophenotyping was performed using the following mAbsto: HLA-C EMR8-5, CD1d CD1d42, CD86 2331, 4-1BBL C65-485, OX40L ik-1,CD3 OKT, Vα24-Jα18 6611, CD4 SK3, CD62L DREG-56, CD134 ACT35, CD1374B4-1, PD-1 EH12.1, GATA3 L50-823 (BD Biosciences), LAG-3 Polyclonal,TIM-3 344823 (R&D System), and rabbit anti-LEF1 EP2030Y mAb (ABCAM). BDor R&D-suggested fluorochromeand isotype-matching Abs were used asnegative controls. The expression of CAR.CD19 on NKTs was determinedusing anti-Id (clone 136.20.1) CD19-CAR specific mAb (Torikai et al.,2013) and goat anti-mouse IgG (BD Biosciences). The analysis wasperformed on a LSR-II five-laser flow cytometer (BD Biosciences) usingBD FACSDiva software v.6.0 and FlowJo 7.2.5 (Tree Star).

In vitro cytotoxicity assay. The cytotoxicity of parental and CAR.CD19NKTs against DAOY or Raji cells was evaluated using 4-hour LuciferaseAssay as previously described (Liu et al., 2013).

Gene expression analysis. Total RNA was collected using TRIzol reagent(Qiagen). Gene expression analysis was performed using Immunology Panelv.2 (NanoString) at BCM Genomic and RNA Profiling Core using nCounterAnalysis System. Data were analyzed using nSolver 2.0 software(NanoString).

In vivo experiments. The colony of NSG mice was originally obtained fromThe Jackson Laboratory and maintained at BCM Animal Care facility. Tumorgrowth was initiated by i.v. injection of 2×10⁵ luciferase-transducedRaji lymphoma cells. On day 3, mice were treated with 4-8×10⁶ CAR-NKTsfollowed by i.p. injection of IL-2 (1000 U/mouse) every 3 days. Tumorgrowth was assessed twice per week by bioluminescent imaging (SmallAnimal Imaging Core facility, Texas Children's Hospital). For the invivo persistence experiments, NKTs were co-transduced with CAR.CD19 andluciferase using retroviral constructs, i.v. injected to tumor-free ortumor-bearing mice, and monitored using bioluminescent imaging twice perweek. Animal experiments were performed according to IACUC approvedprotocols.

Statistics. For in vitro and in vivo experiments, the inventors used2-sided, paired Student's t test to evaluate continuous variable of 2groups, and one-way ANOVA with post-test Bonferroni to evaluatecontinuous variables of more than 2 groups. Survival was analyzed by theKaplan-Meier method and the Log-rank (Mantel-Cox) test to compare pairsof groups. Statistics were computed using GraphPad™ Prism 5.0 (GraphPadSoftware). Differences were considered significant when the p value wasless than 0.05.

Study approval. The cord blood units were obtained from MD AndersonCancer Center Cord Blood Bank according to the protocols approved by theInstitutional Review Boards at MD Anderson Cancer Center (H-16320) andBaylor College of Medicine (H-20911). Written informed consent wasreceived from all participating women prior to inclusion in the studyunder the protocol H-16320. Cord blood units not suitable for clinicaluse (usually due to low cell counts) were either discarded or used forresearch purposes under the protocol H-20911 at Baylor College ofMedicine. Animal experiments were performed according to the protocolAN-5194 approved by the Institutional Animal Care and Use Committees ofBaylor College of Medicine.

Example 4—Effect of IL-21 on NKT Cells

This example demonstrates that IL-21 has a beneficial effect on NKTcells upon exposure of the cells to IL-21. FIGS. 10A-10C show that IL-21increases the frequency of CD62L+ NKT cells during primary expansion.NKTs were isolated from three donors and stimulated with αGalCer-loadedPBMCs and IL-2 (100 U/ml) or IL-2 with IL-21 (10 ng/ml) for 12 days.NKTs were collected and stained for CD62L followed by FACS analysis.FIGS. 11A-11C demonstrates that IL-21 increases the frequency of CD62L+NKT cells during secondary expansion. Following primary expansion (FIGS.10A-10C), NKTs from three donors were re-stimulated with αGalCer-loadedPBMCs and IL-2 (100 U/ml) or IL-2 with IL-21 (10 ng/ml) for 12 days.NKTs were collected and stained for CD62L followed by FACS analysis.FIG. 12 shows expression of CD1d and co-stimulatory molecules on Ramoscells. Ramos B-cell lymphoma cell line was obtained from ATCC andanalyzed by FACS for expression of CD1d, CD86, 4-1BBL, and OX40L usingcorresponding mAbs or IgG control. Ramos cells can expand primary NKTswith high-level CD62L expression (FIGS. 13A-13C). NKTs were isolatedfrom three donors and stimulated with αGalCer-loaded Ramos cells(2×10⁶/well) in IL-2 containing media for 10 days. NKTs were collected,counted and stained for CD3, 6611 and CD62L. Finally, in FIG. 14 it isshown that Ramos cells expand NKTs upon secondary stimulation withsignificant retention of CD62L expression. Following primary expansionwith PBMC (as in FIGS. 10A-10C), NKTs (1×10-/well) were re-stimulatedwith αGalCer-loaded Ramos cells (2×10⁶/well) in IL-2 containing mediafor 10 days. NKTs were collected, counted and stained for CD3, 6611 andCD62L.

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1. A genetically modified natural killer T (NKT) cell comprising at least one chimeric antigen receptor (CAR) expression construct.
 2. The genetically modified NKT cell of claim 1, wherein the NKT cell is CD62L-positive.
 3. The genetically modified NKT cell of claim 1, wherein the NKT cell is a Type 1 NKT cell.
 4. The genetically modified NKT cell of claim 2, wherein the NKT cell is isolated from an enriched population of CD62L-positive NKT cells.
 5. The genetically modified NKT cell of claim 1, wherein the CAR expression construct comprises an antigen recognition domain directed to at least one tumor-associated antigen.
 6. The genetically modified NKT cell of claim 5, wherein the tumor-associated antigens are selected from the group consisting of melanoma-associated antigen (MAGE), expressed antigen of melanoma (PRAME), CD19, CD20, CD22, K-light chain, CD30, CD33, CD123, CD38, CD138, ROR1, ErbB2, ErbB3/4, EGFr vIII, carcinoembryonic antigen, EGP2, EGP40, HER2, mesothelin, TAG72, PSMA, NKG2D ligands, B7-H6, IL-13 receptor a2, MUCI, MUC16, CA9, GD2, GD3, HMW-MAA, CD171, Lewis Y, G250/CAIX, HLA-AI MAGE AI, HLA-A2 NY-ES0-1, PSCI, folate receptor-a, CD44v6, CD44v7/8, 8H9, NCAM, VEGF receptors, 5T4, Fetal AchR, NKG2D ligands, CD44v6 , GPC3, CSPG4, CEA, or combinations thereof.
 7. The genetically modified NKT cell of claim 6, wherein at least one antigen is CD19.
 8. The genetically modified NKT cell of claim 6, wherein at least one antigen is GD2.
 9. The genetically modified NKT cell of claim 6, wherein at least one antigen is GPC3.
 10. The genetically modified NKT cell of claim 4, wherein the CAR expression construct comprises an ectodomain that includes an antigen recognition domain, and a transmembrane domain that links the antigen recognition domain to the transmembrane domain.
 11. The genetically modified NKT cell of claim 10, wherein the spacer is selected from a group consisting of a CH2CH3 region of immunoglobulin, a hinge region from IgG 1, and at least portions of CD3.
 12. The genetically modified NKT cell of claim 11, wherein the transmembrane domain is CD28.
 13. The genetically modified NKT cell of claim 5, wherein the antigen recognition domain is a single-chain variable fragment (scFv).
 14. The genetically modified NKT cell of claim 1, wherein the CAR expression construct comprises at least a portion of a cytoplasmic signaling domain.
 15. The genetically modified NKT cell of claim 14, wherein the cytoplasmic signaling domain is derived from a T cell receptor CD3zeta-chain.
 16. The genetically modified NKT cell of claim 1, wherein the CAR expression construct comprises a costimulatory endodomain.
 17. The genetically modified NKT cell of claim 15, wherein the costimulatory endodomain is selected from CD28, OX40, 4-1BB, ICOS, CD40, CD30, CD27, or combinations thereof.
 18. The genetically modified NKT cell of claim 17, wherein the costimulatory endodomain is 4-1BB.
 19. The genetically modified NKT cell of claim 17, wherein the costimulatory endodomain is CD28.
 20. The genetically modified NKT cell of claim 4, wherein the CAR expression construct comprises a scFv from a CD19-specific antibody FMC-63 which is connected, via a spacer derived from the IgG1 hinge region, to a transmembrane domain derived from CD8α, and a signaling endodomain sequence of 4-1BB fused with a CD3-zeta-chain.
 21. A composition for immunotherapy in a subject comprising a cell of claim
 1. 22. A genetically modified NKT cell comprising a construct expressing a non-natural receptor for a cancer antigen.
 23. A composition for immunotherapy produced by: enriching a population of NKT cells for Type I, CD62L-positive NKT cells that have been genetically modified to include at least one CAR expression construct; and activating the CD62L-positive NKT cells by (a) stimulating one or more T-cell receptors of the NKT cells; and/or (b) stimulating the NKT cells with one or more ligand for T-cell receptors; and/or (c) costimulating the NKT cells with one or more co-stimulatory receptors with one or more agonistic antibodies from the co-stimulatory receptors; and/or (d) costimulating the NKT cells with one or more cytokines; and/or (e) costimulating the NKT cells with an antigen presenting cell that expresses CD1d and one or more ligands of the one or more costimulatory receptors.
 24. The composition of claim 23, wherein the CAR expression construct is a CD19-specific CAR expression construct.
 25. The composition of claim 23, wherein the CAR expression construct is GD2-specific CAR expression construct.
 26. The composition of claim 23, wherein at least one CAR expression construct comprises the costimulatory endodomain 4-1BB. 